The overall goals of the proposed research are to establish the mechanism of action and control of the dynein ATPase in generating ciliary and flagellar movement. The specific goals of these studies can be divided into four parts: (a) we will work to complete our description of the kinetics and thermodynamics of the ATPase cycle by transient and steady state kinetic analysis of the microtubule activation of the dynein ATPase and by equilibrium binding measurements; (b) we will determine the functions and potential interactions of the three dynein heads by examining the ATPase kinetics of dynein subfragments and attempt to relate those studies to the more complex kinetics observed with the three-headed dynein by computer modeling; (c) we will test possible mechanisms of regulation by exploring the effects of calcium, calmodulin and phosphorylation on each step of the ATPase cycle, especially the reactions involved in microtubule activation of the ATPase; (d) finally, we will work to extend these results to the intact axoneme, by examining the kinetics of the intact axoneme directly, and by determining the effects of well characterized monoclonal antibodies on wave propagation in reactivated flagella. The current work builds upon our previous results and well established methods in examining the structure and ATPase pathway of dynein islated from Tetrahymena cilia. We will use stopped-flow and chemical-quench flow methods for rapid kinetic analysis, 18O-isotope exchange studies to examine the lifetime of intermediates in the reaction pathway, and light and electron microscopy to examine the intact axoneme. These studies are expected to establish the complete pathway and mechanism of control by which the hydrolysis of ATP is coupled to dynein crossbridge interaction with microtubules to produce a force for ciliary movement. The work also provides a basis for analysis of dynein-like ATPases in other microtubule systems such as chromosome movement or the transport of membrane bound particles.